Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13R & alpha;1 and IL-4R & alpha;. This complex is assembled by IL-13 initially binding IL-13R & alpha;1, followed by association of the binary IL-13:IL-13R & alpha;1 complex with IL-4R & alpha;. The receptors are shared with IL-4, but IL-4 initially binds IL-4R & alpha;. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (K-D 40 nM-5.5 & mu;M) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 & mu;M). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13R & alpha;1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4R & alpha;. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful F-19 fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these F-19 containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

Automated summary

In this study, a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 and inhibit downstream IL-13 signaling were identified. The study also revealed a novel allosteric mechanism of inhibition and potential small-molecule drug targets for IL-13.